The long term goal of the proposed research is to reveal the evolutionary mechanisms that have given rise to biological species. Speciation is difficult to study, even with pairs of closely related species, because their divergence and isolation from each other limits the degree to which the genetic basis of their divergence can be studied. The proposed work will be on Drosophila pseudoobscura, D. p. bogotana and D. persimilis, three closely related and frequently studied new world species. It is possible to generate fertile hybrids of these species, so one can map and count genes that are responsible for the traits associated with isolation. Three complementary approaches to the study of species differences will be joined in this work. A dense microsattelite map has been developed for these species, and these markers will be use for mapping genes responsible for isolation traits, such as sterility, hybrid inviability, and mater discrimination. (2) At the same time as these studies proceed, the genomic regions that flank the individual microsattelite loci will be the subject of a thorough comparative DNA sequence study of intraspecific and interspecific and interspecific variation. Together these two studies will show which genes are linked to those that contribute to isolation, and it will show the historical pattern of divergence that has occurred for these same regions. Previous research has shown that these species have a history that includes more gene flow for some genomic regions than for others. This means that natural selection, acting against gene flow, has been a critical part of their speciation and divergence. (3) A third study will also be done to measure the amount of introgression, from one species to another, that can occur for each microsatellite locus. These introgression experiments will be done in the laboratory. In contrast to the mapping of isolation traits, the introgression mapping does not rely on investigator identified phenotypes. The introgression mapping will assess the relative fitness of the marked genomic regions, when introgressed into closely related species. Each of these three components draws on similar speciation research done in the past on these three species, though in the proposed work, the mapping will be on a much finer scale with many more markers than has been used previously. The proposed work will also permit entirely novel comparisons of the three types of information which can be brought together to develop a fuller and more integrated picture of how these species came to diverge and the genetic basis of the evolutionary forces that have kept them distinct.